Project Summary/Abstract The introduction of optogenetic methods for controlling activity in defined subsets of neurons has enabled new insights into the functional roles of anatomically distinct brain regions implicated in drug addiction. However, limitations with existing optogenetic tools have made it difficult to address how local connections within a given brain region enable it to integrate and process multiple inputs from other regions. Here we propose to generate a fundamentally unique type of optogenetic tool for silencing defined sets of synapses within local microcircuits, to ultimately understand how they control addiction-related behaviors. Our approach leverages the ability of presynaptic G protein coupled receptors (GPCRs) to inhibit synaptic transmission. In contrast to existing approaches that use direct optical activation of light-sensing GPCRs, our approach involves optical activation of a separate construct on the postsynaptic side of the synaptic cleft, which in turn activates the presynaptic GPCR. In our two aims, we will develop optogenetic tools capable of trans-cellular GPCR activation using high throughput assays in cell lines, and then use experiments in neurons to refine these tools to enable trans-synaptic control of GPCR activation. We will combine optogenetics with electrophysiological readouts in primary neuronal cultures and brain slices to validate their ability to reversibly inhibit synaptic transmission. Successful completion of the proposal will establish new tools with unprecedented synaptic-resolution control over neurotransmission. This technology will enable new experiments to dissect how key brain regions involved in addiction, like the nucleus accumbens, ventral tegmenetal area, and prefrontal cortex, locally integrate information received from diverse brain regions.